Project Summary Toxoplasma gondii is a widespread apicomplexan parasite that causes severe disease in immunocompromised persons and the congenitally-infected fetus. Repeated cycles of host cell invasion and lysis by the actively dividing form of T. gondii, the tachyzoite, are a significant cause of tissue damage during acute infection. Despite the importance of invasion to the life cycle of the parasite and the pathology of toxoplasmosis, relatively little is known about the tachyzoite proteins that mediate the process. In a smallmolecule- based approach to this question, 24 novel small molecule inhibitors and 6 enhancers of invasion were identified. A subset of these compounds affects the invasion of all apicomplexan parasites tested, including malaria parasites, suggesting that they target conserved components of the apicomplexan invasion machinery. Characterization of the biological effects of the compounds and extensive synthetic chemistry has led to the development of specific hypotheses regarding their mechanism of action; three of these hypotheses will be tested in Aims 1-3. The work has also demonstrated the need for general methods to identify the targets of small molecules that are active against the parasite, i.e., methods that would allow for the identification of many small-molecule/target pairs. Such an approach, coupled with high-throughput screening, would represent a powerful way to identify new druggable targets within the parasite. Aim 4 will address this issue, using Yeast-3-Hybrid (Y3H) methodology. The Specific Aims of the proposal are to: (1) Test the hypothesis that compound 115556 inhibits parasite motility and invasion through modification of myosin light chain (TgMLC1); (2) Test the hypothesis that the enhancement of invasion caused by compound 112762 occurs through an effect on parasite protein arginine methyltransferase activity; (3) Test the hypothesis that compound 144146 inhibits conoid extension and invasion through an effect on the parasite protein peroxiredoxin2; and (4) Develop and use Yeast-3-Hybrid (Y3H) methods to identify targets of T. gondii invasion inhibitors This work has the potential to provide important new insights into the proteins and pathways involved in host cell invasion by T. gondii. The results are also likely to be transferable to other, less experimentally accessible human parasites, such as Plasmodium and Cryptosporidium. A major strength of the proposal is the extent to which biological experiments and synthetic chemistry will be integrated to address the project?s goals.